Intracellular Entities Research Articles

Signal Transduction Mediated by Adhesion of Human Trabecular Meshwork Cells to Extracellular Matrix

In this study we investigated the signaling event induced by adhesion of human trabecular meshwork (TM) cells to extracellular matrix (ECM) elements such as fibronectin. The role of tyrosine phosphorylation in adhesion was evaluated. A number of intracellular entities involved in the adhesion-mediated pathways were identified. For the experiments, human TM cells were seeded onto fibronectin- or polylysine (negative control)-coated plates. Fifteen, 30, 90 and 240 min after the seeding, cell lysates were collected. Immunoblotting analysis revealed that tyrosine phosphorylation occurred within 15 min of adhesion of TM cells to fibronectin and the level increased with time. The phosphotyrosyl proteins had molecular masses 25– 220 kDa. A much lower level of tyrosine phosphorylation was observed when cells were plated on polylysine. Immunoprecipitation experiments indicated that the phosphotyrosine-containing proteins included focal adhesion kinase, paxillin, phosphatidylinositol 3-kinase and mitogen activated protein kinase. Within 30 min of adherence to fibronectin, human TM cells immunostained for paxillin and phosphotyrosine and exhibited prominent focal contacts. When treated with tyrosine kinase inhibitors genistein and herbimycin A and a protein kinase C (PKC) pseudosubstrate peptide inhibitor, cell adhesion to fibronectin was compromised and focal contact formation was limited. These results demonstrated that in human TM cells, tyrosine kinase was activated upon their adherence to fibronectin. PKC also appeared to play a role in modulation of the cell-matrix adhesion process. The current study provides insight into the signaling pathways that are linked to the ECM-induced events in TM cells. Elucidation of the hierarchy of signal responses may help develop strategies manipulating the cell-matrix interactions in the TM system.

Immunochemical studies on the photoactive subcellular particles from chromatium

Specific antisera have been prepared in rabbits by injection of pigmented particles isolated by differential centrifugation from extracts of the photosynthetic bacterium Chromatium. These antisera contain antibodies which precipitate the bacteriochlorophyll-containing particles specifically, and can therefore be defined as chromatophore antibodies. As a consequence, analysis of this immune system depends in no way upon the necessity of having a purified preparation of pigmented particles, since precipitation of the pigment-bearing macromolecule by antiserum can be measured independently of any other antigenically reacting species present in particle preparations as colorless contaminants. Most of the antisera however, were found by all criteria to contain only antibodies to pigmented particles, which indicated that the differential centrifugation technique used was effective in separating the pigmented particles from other cell antigens. Absorption of such antisera with intact Chromatium cells removes approximately 50% of the chromatophore antibodies. Absorption of antisera with a surface polysaccharide obtained from the cell by washing removes similar amounts of chromatophore antibodies. These results indicate that the pigment complex of Chromatium is either built into the cell wall or contains components structurally similar to it. These components are polysaccharides. In addition to their relationship to the surface of the Chromatium cell, the pigmented macromolecules contain other antigens which are related immunologically to colorless soluble fractions of the cell. Analogous to the cell surface reactions, these relations can be identified experimentally because the antigen studied is a colored macromolecule. The antigenic interrelationships between the pigmented particles, an identified cell surface polysaccharide, and internal cellular components indicate that the photoactive subcellular particles are not unique intracellular entities but are derived from a more complex cellular structure. The previous chemical and present immunochemical studies suggest that the bacteriochlorophyll in Chromatium may reside in a cephalin-type phospholipide constituting a large part of the cytoplasmic membrane of the cell and that the photosynthetic apparatus is attached to or contained in the wall by a polysaccharide component.